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Abstract. – OBJECTIVE: Wnt/β-catenin path-way plays a critical role in modulating embryon-ic development, cell growth, and differentiation. The over-expression of β-catenin activates this pathway and up-regulates expression of matrix metalloproteinase-13 (MMP-13), and promotes matrix degradation and occurrence of osteoar-thritis (OA). This study aims to explore the effect of miR-320 expression in OA chondrocyte and underlying mechanisms.

PATIENTS AND METHODS: Chondrocyte tis-sues from OA patients and normal individuals were collected for the detection of expression levels of miR-320, β-catenin, MMP-13, and al-pha-1 chain of type II collagen (COL2A1). Dual luciferase reporter assay was performed to test targeted regulation between miR-320 and β-cat-enin. IL-1β was used to simulate in vitro cultured chondrocytes, which were transfected with miR-320 mimic and/or si-β-catenin, followed by quan-tification of miR-320, β-catenin, MMP-13, and COL2A1.

RESULTS: In chondrocytes of OA patients, ex-pression of microRNA (miR)-320 is decreased. Bioinformatics analysis revealed complementa-ry binding sites between miR-320 and β-caten-in. Compared to control group, increasing lev-els of β-catenin and MMP-13 expression with reduction of miR-320 and COL2A1 expressions were observed in OA chondrocytes. Transfec-tion of miR-320 mimic and/or si-β-catenin de-pressed expression of β-catenin and MMP-13 in-side chondrocytes, accompanied with elevation of COL2A1 expression.

CONCLUSIONS: MiR-320 expression in OA chondrocyte is decreased, accompanied with up regulation of β-catenin and MMP-13. MiR-320 can inhibit β-catenin and MMP-13 expressions, elevates COL2A1 expression, which provides novel insights for the treatment of osteoarthritis.

Key Words:MicroRNA-320, Wnt/β-catenin, MMP-13, Osteoar-

thritis.

Introduction

Osteoarthritis (OA), also named as degene-rative arthritis or aged arthritis, represents a type of chronic degenerative bone joint disease featured with degradation of joint cartilage or attachment site of joint boundary ligament, and reactive bone hyperplasia or osteophyte for-mation that can be related with aging, obesity, and trauma1,2. Canonical Wnt/β-catenin signal pathway was firstly recognized in embryonic formation and development. Its participation in regulation of multiple biological processes includes cell growth, differentiation, inflam-matory immunity, and tissue repair3. Previous finding indicated its correlation with functio-nal stability of bone or chondrocyte tissues4. Wnt/β-catenin signal pathway has been shown to be essential for human osteoblast develop-ment, as potentiation of its activity can incre-ase osteoblastic activity, whilst over-activation could destruct chondrocyte tissues for inducing OA5. The over-expression of β-catenin leads to over-activation of Wnt/β-catenin pathway, and plays a significant role in up-regulating expres-sion of matrix metalloproteinase-13 (MMP-13)6,7 or facilitating degradation of chondrocyte extracellular matrix (ECM) or inducing OA8. MicroRNA is one small molecule of non-co-ding RNA with 21-24 nucleotides in eukaryo-tes. It can bind with 3’-untranslatied region (3’-UTR) of target gene mRNA via complete or incomplete binding manners9. Meng et al10 showed significantly lowered miR-320 expres-sion in OA patient’s chondrocytes, indicating the role of miR-320 down-regulation in OA pa-thogenesis. We investigated if miR-320 played a role in regulating OA pathogenesis and the related mechanism.

European Review for Medical and Pharmacological Sciences 2018; 22: 5828-5835

H.-X. ZHANG1, C. SUN1, H.-C. YU1, B. SONG2, Z.-X. PAN1

1Department of Joint Surgery, The No. 89 Hospital of the People’s Liberation Army of China, Weifang, Shandong, China2Information Engineering Department, Weifang Vocational College, Weifang, Shandong, China

Corresponding Author: Zhaoxun Pan, MD; e-mail: b09473162167358@sina.com

Targeted inhibition of β-catenin by miR-320 and decreased MMP-13 expression in suppressing chondrocyte collagen degradation

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Patients and Methods

Clinical InformationA total of 48 OA patients who received whole

knee joint replacement surgery in the No. 89 Ho-spital of the People’s Liberation Army of China from December 2015 to June 2016 were recruited. Tibia samples were collected during the surgery. All cases fitted diagnostic guideline of OA as sti-pulated by American Rheumatology Society, in-cluding: (1) Persistent knee joint pain within one month; (2) X-ray showed formation of boundary osteophyte; (3) Clear and thick joint fluid in at least two assays, with <2000 WBC per mL; (4) Age ≥ 40 years; (5) Duration of morning stiff-ness ≤ 30 min; (6) Sound of friction during joint movement. Knee OA can be diagnosed when sa-tisfying (1)(2), or (1)(3)(5)(6), or (1)(4)(5)(6). OA caused by infection, tumor or rheumatoid disease were excluded. OA patients were classified into grade I (N=25) and grade II (N=23) based on Kel-lgren-Lawrence imaging guideline. Another 20 patients who received post-traumatic amputation were recruited as the control group to collect ti-bia tissues. Patients with diabetes or tumors were excluded. The collection of all tissue samples obtained informed consents from patients. This study has been reviewed and approved by the Ethical Committee of the No. 89 Hospital of the People’s Liberation Army of China.

Major Reagent and MaterialsDulbecco’s Modified Eagle’s Medium (DMEM)

/F12 culture medium, fetal bovine serum (FBS), penicillin-streptomycin, 0.25% trypsin, TRIzol, and type II collagenase were purchased from Gib-co (Rockville, MD, USA). X-tremeGENE siRNA transfection reagent was from Roche (Basel, Swi-tzerland). ReverTra Ace quantitive Reverse Tran-script-Polymerase Chain Reaction (qRT-PCR) kit and SYBR dye was obtained from Toyobo (Osaka, Japan). MiR-320 nucleotide fragment was designed and synthesized by Ruibo Bio (Beijing, China). Rabbit anti-β-catenin polyclonal antibody, mouse anti-MMP-13 monoclonal antibody, rabbit anti-CO-L2A1 polyclonal antibody, and rabbit anti-β-actin polyclonal antibody were bought from Abcam (Cambridge, MA, USA). Horseradish peroxidase (HRP) conjugated secondary antibody was acquired from Zhongshan Jinqiao (Shanghai, China). IL-1β was provided from Santa Cruz Biotechnology (San-ta Cruz, CA, USA). Dual-luciferase reporter assay system and pGL3-promoter plasmids were all pur-chased from Promega (Madison, WI, USA).

Separation and Culture of ChondrocytesCollected cartilage tissues were transferred to

bio-safety cabinet. Chondrocytes were separated under sterile conditions. In brief, cartilage tissues were cut into pieces with 1-3 mm3 size, and were mixed with 0.25% trypsin. After digestion at 37°C for 30 min, tissues were further digested in 0.2% type II collagenase at 37°C for 2.5 h. Tissue debris were filtered out by cell mesh. Chondrocytes were re-suspended in DMEM/F12 culture medium containing 10% FBS and 1% streptomycin-peni-cillin, and were cultured in a chamber with 5% CO2 at 37°C. Culture medium was changed every 2-3 days. Experiments were performed when cells reached 70-80% confluence.

Luciferase Reporter Gene Plasmid Construction

Using human embryonic kidney (HEK) 293 ge-nomic DNA as the template, full-length fragment of wild type or mutant forms of 3’-UTR of β-cat-enin gene was amplified and were cloned into pGL-3M plasmid. Recombinant plasmid was then used to transform DH5α competent cells. Positive clones with correct sequences were screened out by sequencing and were named as pGL3-β-caten-in-3’UTR-wt and pGT-β-catenin-3’UTR-mut.

Luciferase Reporter Gene AssayX-tremeGENE was used to co-transfect

HEK293 cells with 100 ng pGL3-β-catenin-3’UT-R-wt plasmid (or pGL-3-β-catenin-3’UTR-mut), and miR-320 mimic. After 48 h continuous incuba-tion, dual-luciferase assay was performed. In brief, culture medium was discarded. Cells were washed in phosphate buffer solution (PBS) with the addi-tion of 100 μL Passive Lysis Buffer. After 15 min culture, the mixture was centrifuged at 1000 rpm for 5 min. 50 μL cell lysate was mixed with 50 μL luciferase substrate. Activity of luciferase was me-asured immediately. The enzymatic reaction was stopped in 50 μL Stop & Glo, followed by quantifi-cation of sea pansy luciferase activity. The relative expression level of reporter gene was calculated as the ratio of luciferase activity against sea pansy luciferase activity. Oligonucleotide sequences used were: scramble NC, 5’-UUCUC CGAAC GU-GUC ACGUU U-3’; miR-320 mimic, 5’-AAAAG CUGGG UUGAG AGGGC GA-3’.

Transfection and Grouping of Chondrocytes

In vitro cultured chondrocytes from OA patien-ts were treated with 10 ng/mL IL-1β for 48 h. Cel-

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ls were assigned into five groups: scramble NC transfection group; miR-320 mimic transfection group; si-NC transfection group; si-β-catenin group; and miR-320 mimic + si-β-catenin group. Nucleotide fragments used were: si-β-catenin sense: 5’-UGGUU GCCUU GCUCA ACAA-3’; si-β-catenin anti-sense: 5’-ACCAA CGGAA CGAGU UGUU-3’; si-NC sense: 5’-UUCUC CGAAC GUGUC ACGUU U-3’; si-NC anti-sen-se, 5’-ACGUG ACACG UUCGG AGAAU U-3’.

qRT-PCR for Gene Expression AssayCartilage tissues were homogenized in liquid

nitrogen. TRIzol reagent was added to lyse cel-ls. After layering, RNA precipitation, elution and re-suspension, RNA was obtained. ReverTra Ace qPCR RT Kit synthesized cDNA from RNA by reverse transcription. Using cDNA as the templa-te, PCR amplification with the addition of SYBR fluorescent dye. Data were collected for analysis. PCR conditions were: 95°C for 15 s, followed by 60°C 30 s and 74°C 30 s. 40 cycles were perfor-med on ABI ViiA TM7 fluorescent PCR cycler (Waltham, MA, USA). Primer sequences used for PCR were: miR-320PF: 5’-ACACT CCAGC TGG-GA AAAGC TGGGT TGAGA-3’; miR-320PR: 5’-ACACT CCAGC TGGGT CGCCC TC -3’; U6PF: 5’-ATTGG AACGA TACAG AGAAG ATT-3’; U6PR: 5’-GGAAC GCTTC ACGAA TTTG-3’; β-cateninPF: 5’-AGGAC CACCG CATCT CTACA T-3’; β-cateninPR: 5’-GCAGT TTTGT CAGTT CAGGG A-3’; COL2A1PF: 5’-TGGAC GCCAT GAAGG TTTTC T-3’; COL2A1PR: 5’-TGGG AGCCA GATTG TCATC TC-3’; MMP-13PF: 5’-CCAGA CTTCA CGATG GCATTG-3’; MMP-13PR: 5’-GGCAT CTCCT CCATA ATTTG GC-3’; β-actinPF: 5’-GAACC CTAAG GCCAA C-3’; β-actinPR: 5’-TGTCA CGCAC GATTT CC-3’.

Western BlotCartilage tissues were mixed with homogeni-

zing buffer for tissue lysate. Protein supernatant was prepared after 12 000 rpm centrifugation for 10 min. Bovine serum albumin (BSA) approa-ch was used to test protein quantity and quality. After boiling for 5 min in 4X loading buffer, 50 μg protein samples were separated in 10% sodium dodecyl sulfate-polyacrylamide gel electrophore-sis (SDS-PAGE) (2.5-3 h), and were transferred to polyvinylidene difluoride (PVDF) membrane in a Bio-Rad (Hercules, CA, USA) Wet transfer cham-ber (300 mA current, 90 min time). The membra-ne was blocked in 5% defatted milk powder for 60 min, followed by primary antibody (anti-β-caten-

in at 1:400, anti-MMP-13 at 1:200, and COL2A1 at 1:200 or anti-β-actin at 1:500) incubation at 4°C for 12 h. By Phosphate-Buffered Solution Twe-en-20 (PBST) washing (3 times), HRP-labeled se-condary antibody (anti-mouse or anti-rabbit at 1:8 000 dilution) was added for 60 min incubation un-der room temperature. After PBST rinsing three times, electrochemiluminescence (ECL) reagent was added for 1-3 min incubation in the dark. The membrane was then exposed in the dark and scan-ned for data analysis using Quantity One software (Bio-Rad, Hercules, CA, USA).

Statistical AnalysisSPSS18.0 (SPSS Inc., Chicago, Il, USA) was

used for data analysis. Measurement data were presented as mean±standard deviation (SD). The Student t-test was used to compare measurement data between groups. Differences between mul-tiple groups were compared using analysis of variance with Tukey’s post-hoc test. A statistical significance was defined when p<0.05.

Results

Reduced MiR-320 and Elevated β-Catenin Expressions in Cartilage of OA Patients

qRT-PCR results showed that miR-320 expres-sion in cartilage tissues of OA patients was signi-ficantly reduced compared to those in controlled cartilage tissues (p<0.05) (Figure 1A), whilst β-catenin mRNA level was statistically increased (p<0.05) (Figure 1B). Moreover, in OA patients, MMP-13 mRNA level in their cartilage tissues was significantly elevated compared to that in control tissues (p<0.05) (Figure 1C). Conversely, COL2A1 mRNA expression was statistically de-clined (p<0.05) (Figure 1D). Western blot obtai-ned consistent results that protein levels of β-cat-enin and MMP-13 in OA cartilage tissues were remarkably elevated (Figure 1E), whilst COL2A1 protein was decreased (Figure 1E). These results suggested that down-regulation of miR-320 with rise of β-catenin were correlated with OA onset.

miR-320 Targeted and Inhibited β-Catenin Expression

Online prediction of the target gene by microR-NA.org showed the targeted binding site between miR-320 and 3’-UTR of β-catenin (Figure 2A). Transfection of miR-320 mimic significantly de-creased relative luciferase activity in HEK293 cells previously transfected with pGL3-β-caten-

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in-3’-UTR-wt plasmid (p<0.05). The relative luci-ferase activity in HEK293 cells transfected with pGL3-β-catenin-3’-UTR-mut plasmid, however, was not changed by miR-320 mimic transfection (p>0.05). The transfection of scramble NC had no

significant effects on relative luciferase activity in HEK293 cells expressing both plasmids (p>0.05). These results indicated that miR-320 could speci-fically targeted on 3’-UTR of β-catenin-gene and regulated its expression (Figure 2B).

Figure 1. Lower miR-320 and elevated β-catenin expressions in cartilage tissues of OA patients. A, qRT-PCR for miR-320 expression. B, qRT-PCR for β-catenin mRNA. C, qRT-PCR for MMP-13 mRNA. D, qRT-PCR for COL2A1 mRNA. E, We-stern blot for protein expression.

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MiR-320 Up-Regulation Inhibited Expression of MMP-13 and β-Catenin in Chondrocytes

Transfection of miR-320 mimic and/or β-cat-enin siNRA significantly lowered MMP-13 and β-catenin expression in chondrocytes (p<0.05) (Figure 3A and 3B). Meanwhile, COL2A1 expres-sion was remarkably elevated (Figure 3A and 3B), indicating that miR-320 up-regulation could suppress Wnt/β-catenin signal pathway activity, down regulate MMP-13 expression, and weaken the inhibitor effects on COL2A1throuhg increa-sing COL2A1 expression.

Discussion

OA is a type of degenerative cartilage joint di-sease. In aged people, fewer feeding vessels un-der cartilage cause lower elasticity, friction and structural destruction as a result of disorders in synthesis and metabolism of extra-cellular matrix of cartilage tissues11. OA is featured as degene-rative change of joint cartilage and subchondral bones, joint pain, difficulty in activity, and oste-ophyte formation in clinics, with aged people as the major patient population12. OA has relatively higher incidence worldwide. It is estimated that the overall OA incidence was 15%, with 20-30% increments by every 10 years age13. There have

been currently about 150 million OA patients in China, leading to severe influences on labor ca-pability and life quality14. The morbidity of OA reaches 53% eventually, forming a major reason of working ability deprivation and mobility disor-der15,16. The core pathological change of OA inclu-des defects in joint cartilage and osteophyte for-mation, both of which are results under combined effects including imbalance of catabolic/anabolic metabolism of joint chondrocytes and cartilage matrix, and focal inflammatory response inside joint cavity, resulting in joint swelling/pain, acti-vity restriction and joint malformation17-19. OA is implicated with a complicated pathogenesis, al-though age is strongly involved, other factors still contribute significantly to OA onset.

Canonical Wnt/β-catenin signal pathway is cor-related with tissue/organ formation, cell growth/differentiation20. Canonical Wnt/β-catenin signal pathway consists of Wnt protein, transmembrane protein frizzled (Frz), co-receptor LRP5/6, di-sheveled protein (Dsh), axin, glycogen synthase kinase-3β (GSK-3β), adenomatous polyposis coli (APC), β-catenin, T-cell factor/lymphoid enhan-cing factor (TCF/LEF)21. In the absence of Wn-t/β-catenin signal pathway activating factors, β-catenin can form complexes with Axin, APC, and GSK-3β. It is then phosphorylated by CK1 and GSK-3β, and free cytosolic GSK-3β level is decreased. When Wnt/β-catenin signal pathway is activated, Wnt protein binds with extracellular domain of Frz protein. It further recruits and acti-vates Dsh, denatures the axin-GSK-3β-APC-β-catenin complex, thus eliminates phosphorylation between β-catenin and CK1 or GSK-3β. β-catenin accumulates inside cytoplasm, followed by nucle-ar translocation, and binds with transcriptional factor TCF/LEF to facilitate transcription and expression of downstream target genes22.

Matrix metalloproteinases (MMPs) belong to proteinase superfamily with important roles in degradation of extracellular matrix, and they are involved in important physiology and patholo-gy processes including embryonic development, osteogenesis and cartilage development23, and tu-mor invasion or metastasis24. MMPs mainly con-sists of collagenase (MMP-1, -8 and -13), matrix lyse (MMP-3, -7 and -11) and gelatinase (MMP-2, -9). Previous findings showed that abnormally elevated MMPs in focal region of cartilage tissues is one critical reason causing imbalance in syn-thesis and degradation of extracellular matrix. MMP-13 represents a potent enzyme specifically targeted on type II collagenase, which is the most

Figure 2. MiR-320 targeted and inhibited β-catenin expression. A, Binding sites between miR-320 and 3’-UTR of β-catenin mRNA. B, Dual luciferase reporter gene assay.

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featured and abundantly distributed protein insi-de cartilage matrix, thus degrading extracellular matrix. Moreover, other subtypes of MMPs can only exert degrading effects on type II collagen via MMP-1325. A previous study26 indicated that degradation on major component of joint cartila-ge, type II collagen, by MMP-13 contributed to OA onset. The over-activation of Wnt/β-catenin signal pathway plays important roles in up-regu-lating MMP-13 expression6,7 and facilitating de-gradation of cartilage matrix and inducing OA8.

Results from this study showed significantly lower miR-320 expression level in cartilage tis-sues of OA patients compared to those in control group. Meng et al10 also found lower miR-320 expression in OA patient’s cartilage, as consi-stent with our study. Compared to control group, OA cartilage tissues also showed remarkable elevation of β-catenin and MMP-13 expressions, whilst COL2A1 expression was suppressed, in-dicating potential role of β-catenin up-regulation in enhancing MMP-13 expression, degrading COL2A1 and facilitating OA pathogenesis. Zhou et al27 found similar results showed higher β-cat-enin and MMP-13 levels in OA cartilage tissues. Liu et al28 revealed over-activation of Wnt/β-cat-enin signal pathway and elevated MMP-13 expression in cartilage tissues from OA model rats. Zhu et al8 reported significantly enhanced MMP-9 and MMP-13 expression in cartilage of transgenic mice with β-catenin over-expression,

accompanied with smaller cartilage tissue area and collagen precipitation, OA-like pathogene-sis in knee joint tissues. This study indicated the possible correlation between β-catenin and MMP-13 up-regulation in cartilage tissues with OA onset, probably sharing common mechani-sms with Zhou et al27 and Liu et al28. This work revealed that the inhibition of β-catenin signal molecules could decrease MMP-13 expression inside cultured chondrocytes, thus suppressing its degradation effects on type II collagen, incre-asing COL2A1 expression, supporting the result by Tamamura et al29. Zhou et al27 also showed that Tetrandrine could inhibit activity of Wn-t/β-catenin signal pathway and MMP-1 expres-sion in cultured chondrocytes under stimulus by IL-1. Importantly, our investigation showed an alternative basis for the regulation of OA with miRNAs besides the inhibitory role of miR-138 via targeting p6530.

Conclusions

We demonstrated that miR-320 expression was reduced in cartilage tissues of OA patients, with high expression of β-catenin and MMP-13. MiR-320 can inhibit β-catenin and MMP-13 expres-sions, and elevates the level of COL2A1, which provides fundamental scaffold for the therapy of OA in the future.

Figure 3. Elevation of miR-320 expression inhibited β-catenin and MMP-13 expression. A, qRT-PCR for gene expression. B, Western blot for protein expression. *, p<0.05 comparing between miR-320 mimic and scramble NC groups; #, p<0.05 compa-ring between si-β-catenin and si-NC groups; ∆, p<0.05 comparing between miR-320 mimic + si-β-catenin and Scramble NC groups; &, p<0.05 comparing between miR-320 mimic + si-β-catenin and si-NC group.

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Conflict of InterestThe Authors declare that they have no conflict of interest.

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